Methods 50 (2010) 302–307
Contents lists available at ScienceDirect
Methods
journal homepage: www.elsevier.com/locate/ymeth
Review Article
Circulating nucleic acids in cancer and pregnancy
Pamela Pinzani *, Francesca Salvianti, Mario Pazzagli, Claudio Orlando
Department of Clinical Physiopathology, University of Florence and Istituto Toscano Tumori, Viale Pieraccini 6, 50139 Florence, Italy
a r t i c l e
i n f o
Article history:
Accepted 5 February 2010
Available online 8 February 2010
Keywords:
Cell-free DNA
Cell-free RNA
Tumors
Plasma
Serum
a b s t r a c t
Circulating nucleic acids are present in the blood of humans and other vertebrates. During the last 10 years
researchers actively studied cell-free nucleic acids present in plasma or serum with great expectations of
their use as potential biomarkers for cancer and other pathologic conditions. In the present manuscript
the main findings related to the principal characteristics of circulating nucleic acids, the hypothesis on their
origin and some methodological considerations on sample collection and extraction as well as on some
innovative assay methods have been summarized. Recent reports on the importance of circulating nucleic
acids in the intercellular exchange of genetic information between eukaryotic cells have been reviewed.
Ó 2010 Published by Elsevier Inc.
1. General issues on circulating nucleic acids
Nucleic acids circulating in human blood but also in other body
fluids (lymphatic fluid, liquor, ascites, milk, urine, stool and bronchial lavage) can be distinguished as either cell-free (CF) or cellassociated nucleic acids.
Circulating nucleic acids (CNA) have been firstly identified by
Mandel and Metais in 1948 [1], but no association with disease
was hypothesized. Only 30 years later, in 1977, Leon et al. [2]
found CNA in plasma of patients affected by lung cancer.
Different hypotheses were done to explain the origin of CNA.
They are supposed to derive from cells dead for necrosis or apoptosis [3], but some authors reported the possibility of an active release from cells [4–8]. The presence of DNA and RNA circulating
freely in the blood stream of healthy subjects can be related to activated lymphocytes and to the lysis of other nucleated cells or to
their active secretion. In patients affected by neoplastic diseases
it is supposed that normal and cancer cells can: (i) detach from
the tumor mass and undergo necrosis or apoptosis; and alternatively (ii) actively release nucleic acids in the blood flow.
The quantity of circulating DNA is generally very low in healthy
subjects (less than 5 ng/ml of plasma), while it increases (5–10
times) when considering subjects affected by a neoplastic disease
[9–12] as well as in some physiologic conditions, such as pregnancy
[13,14]. In physiologic conditions as well as in benign pathologies, CF
DNA seems to derive almost entirely from apoptosis of circulating
cells [15]. CF DNA is double stranded and in the form of nucleoproteic
complex. Studies on fetal DNA, reported an half-life of 16.3 min for
CF DNA [16] that can be reasonably extended to circulating DNA in
general as confirmed by experiments conducted by injecting puri* Corresponding author. Fax: +39 0554271371.
E-mail addresses: [email protected], [email protected]fi.it (P. Pinzani).
1046-2023/$ - see front matter Ó 2010 Published by Elsevier Inc.
doi:10.1016/j.ymeth.2010.02.004
fied DNA into the blood stream of animals [17]. Moreover, the pattern of circulating DNA was found to be similar to that of apoptotic
cells when analyzed by gel electrophoresis [18,19] and by sequencing reactions [20–23]. Nevertheless also high molecular weight DNA
fragments have been retrieved [19] and their presence was associated to the necrosis of tumor cells. Results in this field are often in
disagreement. Moreover, all the previous reports regarding the genetic analysis of CF DNA showed that high molecular weight fragments do not contain tumor-specific mutations which on the
contrary are frequent in small DNA circulating fragments, probably
deriving from phagocitated necrotic cells [24].
Differences in the nature of CF DNA may be an interesting pathological indicator, suggesting that in some pathologies, the measurement of DNA integrity may provide a simple and inexpensive
method for cancer detection [3].
As regard to the biological significance of CNA, Garcia-Olmo and
co-workers [25,26] suggested that metastases might develop as a
result of transfection of susceptible cells in distant target organs
with dominant oncogenes that circulate in the plasma and are derived from the primary tumor.
When analyzing the results present in the literature, discordant
results can be attributed to several factors, but the source of discrepancies can be the use of plasma or serum as the starting material. Several anticoagulants (EDTA, heparin, and lithium-heparin)
have been used and, within the same biological fluids, the variability depends upon the use of different protocol for sample processing (i.e. sample collection and nucleic acid extraction procedure).
In particular, little is known about blood sample processing and
especially about the delay in separating the plasma or serum,
variation in centrifugation steps and duration and conditions of
storage [27]. On the contrary all the studies reported the extraction
method used so that the results could be read in the light of the
different procedure utilized. Last but not least, every original article
P. Pinzani et al. / Methods 50 (2010) 302–307
implies a different technique for detecting the CNA, some being
quantitatively oriented, whereas others evaluate only the qualitative aspects. Moreover, even the quantitative real time methods differ in the choice of the template gene and consequently in the assay
characteristics.
Thus, the mechanism and the nature of nucleic acids circulating
in serum and plasma has not been clarified yet, notwithstanding
many articles reported biomarker validations among CNA. Recently, comparison of CNA with genomic DNA by next-generation
sequencing [22] suggests that non specific DNA release is not the
sole origin for CNA and that apoptotic genomic DNA is the major
but not the unique source of CNA in apparently healthy individuals. The authors did not find a circulating DNA pool consisting
purely of unspecific apoptotic or necrotic nuclear DNA, failing in
evidencing classes of sequences differing from circulating and
genomic DNA, apart for the detection of over-represented Alu sequences in serum DNA [22].
The apparent higher content of DNA in serum relatively to
plasma can be explained by the clotting process of blood cells that
can cause nucleic acid release [28,29]. Concordantly with the above
cited papers it was reported that the majority of cell-free DNA in
serum samples is generated in vitro by lysis of white blood cells
[30]. This is the reason why plasma seems more convenient and
adequate for the study of CNA, since its analysis avoids the simultaneous testing of material originally associated to cells, as demonstrated for fetal DNA detection in maternal plasma [31].
2. Updated procedures for sample collection
Relatively to sample collection procedures, it was reported that
both a delay in blood processing and storage temperature can
influence the amount of DNA extracted from plasma [32]. Anticoagulants do not influence the quantity of the recovered DNA from
plasma, but EDTA shows a stabilizing effect on blood during the
time between sample draw and processing, both at room temperature and at 4 °C [32,33].
In order to get rid of contaminating DNA derived from cells, both
filtration [34] and repeated centrifugations [35–37] at low and high
speed were reported, demonstrating that no release of circulating
nucleic acid was induced from blood cells even at maximum centrifugation speed. Regarding the stability of CNAs in the frozen samples, some authors showed that plasma can be conserved frozen
for years (at least 2 for RNA and 6 for DNA) [38–40] at 70 or
20 °C without affecting CNA concentration, while other authors reported a decay of 30% in DNA from stored plasma [29].
In our lab the following procedure was adopted for plasma sample collection:
(i) Samples are collected in EDTA-containing tubes and they
should arrive in lab within 1 h from blood draw.
(ii) They are submitted to a first centrifugation step at 1600g,
4 °C for 10 min. Plasma will be recovered.
(iii) A second centrifugation is performed at maximum speed, at
4 °C, for 10 min. Pellets eventually formed in this step will be
discarded. Plasma will be split into one-extraction-aliquots
of 500 ll each.
(iv) Plasma will be maintained at 80 °C until extracted.
303
The isolation of CF DNA from plasma and serum represents a
challenge, due to its small quantity and fragmented nature [42].
A recent study [43] compares different commercial kits for DNA
isolation (QIAamp DNA Mini Blood Kit by Qiagen, Agencourt Genfind Blood and Serum Genomic DNA Isolation Kit by Agencourt Bioscience Corporation, QIAamp Virus Spin Kit by Qiagen, Invitrogen
ChargeSwitch gDNA 1 ml Serum Kit by Invitrogen) reaching the
conclusion that the QIAamp Virus Spin Kit gives the best yields
from serum and plasma. Other authors [42] reported the superiority of the NucleoSpin Plasma XS Kit by Macherey–Nagel in terms of
DNA yield, purity, and retrieval of small DNA fragments with respect to the QIAamp DNA Blood Mini Kit (QIAGEN), which is widely
used by investigators in the field of CNA.
In a report on circulating fetal DNA in maternal plasma [44] the
NucliSens Magnetic Extraction system (BioMérieux) and the QIAamp DSP Virus Kit (QIAgen) are compared with QIAamp DNA Blood
Mini Kit (QIAgen). Both methods were assessed to improve total
DNA yield. QIAamp DSP Virus Kit provided the best results for fetal
versus total DNA, as it preferentially extracts small size DNA
fragments.
In our experience the comparison between the two most widely
used kits (DSP and QIAamp) confirmed these findings, as explained
in the section dedicated to the DNA integrity index.
3.2. RNA extraction
Also cell-free RNA (CF RNA) suffers from the same pre-analytical problems encountered for CF DNA, with the peculiar difference
to show higher stability than that expected on the basis of the
characteristics of tissue-extracted RNA.
Thus RNA from plasma survives up to 24 h [45]. Standardized
kits specifically designed for viral RNA extraction (i.e. DSP Virus
Kit or QIAamp UltraSens Virus Kit, Qiagen, Hilden, Germany)
showed appropriate performances for the isolation of CF RNA from
human plasma, using silica gel membrane technology. CF RNA was
reported to be complexed to other molecules, making it resistant to
nucleolytic attacks. Optimized protocols may lead to higher
amounts of CF RNA and this finding confirms the importance of
pre-analytical factors in CNA isolation [46].
4. Circulating DNA parameters
4.1. DNA quantity
The need for a correct quantification of CF DNA was evident
when significant differences between cancer patients and the control population were demonstrated [47,48]. Total plasma DNA concentration is now considered as an unspecific biomarker, but easily
identifiable in neoplastic patients as well as in some benign diseases. Initial methods ranged from diphenylamine staining [49],
Dipstick Kit [50], counter immuno-electrophoresis [51] spectrophotometric measurement [52], picogreen assay [53] and real-time
quantitative PCR (qPCR) [54]. An increased plasma concentration
can be observed at an early stage of tumor development [55] as
well as during the first trimester of pregnancy [56]. This parameter
has been studied extensively [41], but generally a lack of correlation between plasma DNA concentration and tumor size, stage or
location has been reported.
3. Nucleic acid extraction method – CNA extraction
4.2. DNA integrity
3.1. DNA extraction
The extraction method is an important issue to be addressed in
the field of CNA, for which there is no agreement in literature and
several protocols have been reported [41].
DNA integrity is a parameter of interest for plasma CF DNA,
since it could be used to extrapolate the origin of circulating
DNA. As stated in the introduction, the dimension of DNA fragments is related to the mechanism of release from the cells. This
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P. Pinzani et al. / Methods 50 (2010) 302–307
aspect can represent an useful parameter also in the view to clarify
this problem. Few papers compared the DNA integrity index of
cancer patients with that of healthy subjects, generally leading to
the common conclusion that circulating DNA from cancer patients
possesses a higher integrity index than the control population [57–
63]. On the other hand, no difference was found between benign
and malignant tumors [64,65]. The design of the assays differs significantly in regard to the target genes (ALU repeats, hTERT, . . .) but
shares the same principle for assay design, based on the use of a
common forward primer and reverse primers delimitating amplicons of increasing dimension. The ratio between the quantity of
the long versus the short amplicon represents the so called integrity index.
We built up an assay based on the amyloid precursor protein
(APP) gene detection by qPCR, designing four different assays with
comparable efficiency for the amplification of a 67, 180, 306 and
476 bp amplicon, respectively (unpublished data). This procedure
allowed us to compare three integrity index (180/67, 306/67 and
476/67) to be used for the investigation of the circulating DNA
integrity. Just to compare the extraction procedure we considered
only healthy subjects whose plasma samples were submitted to
the two different extraction procedures (DSP Virus Kit and QIAamp
Mini Kit, Qiagen, Hilden, Germany). The results are reported in
Fig. 1, where it can be evidenced that plasma total DNA concentration varies on the basis of the amplicon length. The two kits differ
mainly when fragments between 67 and 306 bp are considered
(Fig. 1). The apoptotic origin of the CF DNA fragments in healthy
subjects seems to be confirmed by the evidenced higher concentration of fragments of dimensions comprised between 180 and
306 bp.
5. Cell-free DNA as a biomarker of cancer or disease
Most reports related to the diagnostic and prognostic applications of CF DNA are relative to neoplastic diseases. Nevertheless,
the increase of circulating CF DNA is not a specific marker for
malignant conditions. Plasma DNA levels are increased also in
other physiological status and diseases, for example pregnancy
and trauma. The detection in plasma/serum DNA of oncogene
mutations, often encountered in a wide variety of cancer tissues,
provides concrete evidence that CF nucleic acids are released into
the circulation by tumors [47]. Thus, methods for the detection
of tumor-specific DNA variants have been developed.
The main limitation to the molecular diagnosis of solid tumors
is the frequent need for invasive procedures to obtain adequate
testing materials [66]. So if, from a theoretical point of view, CNA
are well accepted as non-invasive markers readily available in
any stage of the disease or at any time in the follow up period, their
low concentration in plasma limits their use and represents a way
of selection for the methods to be used for detection and quantification. During the last 10 years there was a growing interest in circulating DNA, as demonstrated by the increasing number of
publications. The analytical requirements of the methods used to
study circulating DNA are so demanding for a strict specificity
and a high sensitivity that the ideal method is still far to be found.
Obviously many attempts are ongoing in designing new methods
able to overcome the sensitivity limitation of conventional PCR
and sequencing methods, but the number of publications already
available in the field is so large that representative papers will be
chosen for discussion. The mutation analysis on DNA extracted
from plasma was related to the detection of variants in the K-ras
oncogene. In fact it was chosen as a tumor specific marker, since
it shows a very high mutation frequency. K-ras mutations are precocious events in neoplastic development starting from codons 12,
13 and 61 and detectable with high specificity and sensitivity in
patients affected by colorectal cancer [67,68]. It was reported an
83% correspondence between the results found in plasma with
those obtained from tissue [38] thus bringing to the definition of
plasma as a surrogate sample for tumor tissue. Moreover, some patients showed mutated DNA in the CF compartment also in the absence of an evident neoplastic disease and this could represent a
risk factor for tumor development [69]. K-ras DNA in plasma was
studied also for the diagnosis of other cancers. The results did
not show any significant relationship with the development of
non small cell lung cancer (NSCLC), failing to confirm K-ras as a
marker of tumor presence in this cancer type [70]. On the contrary,
K-ras mutations were detected in patients with pancreatic cancer
5–14 months before tumor diagnosis and were absent in patients
with benign pancreas pathologies and in healthy subjects [71].
As a signature of tumor-derived DNA, mutations in oncogenes
and tumor suppressor genes can be used as tumor biomarkers. Besides K-ras mutation, the detection of common p53 mutations in
plasma of smokers without cancer but not in the plasma of nonsmokers indicated that the presence of p53 mutations in plasma
DNA could reflect the exposure to carcinogens and, hence, the
chance of developing lung cancer [72].
Other indicators of the presence of tumor-deriving DNA have
been adopted such as microsatellite alterations [73–75], viral
DNA [76], hypermethylation of tumor suppressor gene [77].
6. Cell-free RNA in cancer
Fig. 1. Cell-free DNA concentration in plasma from healthy donors (n = 15). On the
basis of the amplicon size used by the real-time PCR methods, it was possible to
calculate the amount of cell-free DNA (ng/ml of plasma) of DNA fragments of
dimension comprised in the following ranges: (i) 67–180 bp; (ii) 181–306 bp and
(iii) 307–476. Measurement of DNA concentrations were performed following two
different type of extraction procedures: DSP Virus Kit (white columns) and QIAamp
Mini Kit (black columns).
When dealing with circulating RNA two different aspects must
be considered: RNA can be cell-associated or cell-free.
The significance of mRNA deriving from cells has been widely
investigated in the field of oncology with the aim to detect circulating tumor cells (CTCs). As there are no disease-specific markers uniquely associated with any solid cancer [78], the presence of CTCs
in the blood stream has been assessed by indirect measurements of
tissue-specific transcripts by RT-qPCR (reverse transcription-quantitative real-time PCR). However, the above aspect will not be considered in this paper which is focused on CF RNA.
The presence of CF RNA in plasma has been demonstrated by Lo
et al. [79] as well as Kopresky et al. in 1999 [40]. Since then, the
presence of tumor associated CF RNA has been detected in the
plasma/serum of subjects affected by various cancers such as
P. Pinzani et al. / Methods 50 (2010) 302–307
breast [80], colon [81] and lung [82], hepatocellular [83] and prostatic [84] carcinoma, gastric cancer [85] by qualitative PCR approaches or RT-qPCR [41].
The stability of RNA in plasma has been studied leading to the
discovery that RNA is protected from RNase degradation being particle-associated [86]. These findings have been recently confirmed
by Garcìa et al. [87] who also showed that this RNA fraction is enriched in mRNAs and small RNAs, suggesting a mechanism of active release from cells.
7. Cell-free RNA in pregnancy
The discovery of circulating fetal RNA in plasma of pregnant
women [88] opened new perspectives in the research of new
methods for non-invasive prenatal diagnosis. The source of fetal
CF RNA in maternal circulation is represented by hematopoietic
cells and the placenta [89].
The first detection of placental mRNAs in maternal plasma was
reported by Ng et al. [90] who developed RT-qPCR methods specific
for mRNA transcripts of the genes coding for human placental lactogen (hPL) and the beta subunit of human chorionic gonadotropin
(bhCG).
Placental mRNA markers (hPL, bhCG, corticotropin-releasing
hormone, tissue factor pathway inhibitor 2, KISS1, and placentaspecific protein 1) to be used in non-invasive prenatal gene expression profiling were identified by a micro-array based approach by
Tsui et al. [91] and subsequently confirmed by RT-qPCR.
A preponderance of 50 mRNA fragments in circulating placental
RNA was later observed, with potentially interesting implications
for the development of new markers to be used in prenatal diagnosis [92].
The discovery of the presence of placental CF RNA since the 4th
week of gestation [93] has confirmed the important role that can
be played by this target in prenatal screening.
A great input in the field of placental CF RNA has been given by
the study of markers related to disorders associated to pregnancy,
in particular preeclampsia. Starting from the demonstration of increased levels of corticotropin-releasing hormone (CRH) mRNA in
preeclampsia, compared with non preeclamptic pregnancies
matched for gestational age [94], a series of markers were investigated, such as leptin [95] and urocortin [96].
In the first trimester of pregnancy circulating bhCG mRNA concentrations resulted significantly lower in the serum of mothers
carrying fetuses with trisomy of chromosome 18 than in that of
mothers with euploid fetuses [97]. Another approach to the same
issue was carried out by Lo et al. [98] who investigated trisomy
21 fetuses by assessing the ratio between alleles of a single-nucleotide polymorphism (SNP) in the placenta-specific 4 (PLAC4)
mRNA expressed by the placenta. The relative abundance of the
two SNP alleles in heterozygous fetuses was determined by primer
extension and mass spectrometry [98]. Recently [99] the principles
of digital PCR have been applied to the detection of fetal chromosomal aneuploidy, measuring the allelic ratio of PLAC4 mRNA in
maternal plasma. Digital PCR involves multiple PCR analyses on extremely diluted nucleic acids (one template molecule per reaction
well): the proportion of positive amplifications among the total
number of PCRs analyzed allows an estimation of the template
concentration in the original non diluted sample.
8. An update on methodology
The most diffuse and suitable technique used to study CNA is
PCR in its different applications.
To evidence tumor-associated mutations in the plasma/serum
of cancer patients allele-specific polymerase chain reaction is
305
the method of choice to detect mutated sequence present at a
low concentration among a background of wild-type sequences
[100,101].
This approach will require the design of allele-specific primers
and hence the sequence information of the mutation and it would
miss the tumor-associated variants occurring outside the mutation
hot spots. Moreover, often the sensitivity characteristics of the
method are not sufficient to detect the presence of the mutation
in plasma. Alternative methods can be represented by emerging
mass spectrometry-based method [102], but they do not solve all
the above cited problems. For the identification of low level somatic mutations within wild-type DNA, a new PCR method has
been reported named COLD-PCR [103]. It can be used in conjunction with high resolution melting analysis (HRMA) [104] improving the mutation scanning potency of this technique. It shows an
increase in assay specificity and sensitivity over the conventional
PCR, reaching detection limits of 2% of mutated allele in wild-type
DNA [104].
Among the most useful technologies to be used in this field realtime PCR appears to be one of the best candidates to solve most of
the problems related to post-PCR handling, assay sensitivity and
specificity. In fact, it was reported as a method particularly suited
for plasma mutation detection and quantification, since it reduces
the chance of contamination and it can make use of molecules
enhancing the discriminating power of the assay. For example,
peptide nucleic acid (PNA) clamping and a locked nucleic acid
(LNA) hybrid probe have been used to detect a single point mutation in plasma [105] and LNA oligonucleotides, that competitively
inhibit primer binding to wild-type DNA, were adopted for selective amplification of rare mutations [106].
A COLD-real time approach has also been reported [107] and it
can be reasonably considered up to now one of the techniques with
the highest sensitivity and specificity to be used for rare mutated
species. Anyway, COLD-PCR methods require a big effort for discovering the critical temperature which allows the preferential
amplification of the mutated allele and moreover it can be applied
to the detection of known variants.
9. Intercommunication via circulating nucleic acids
The intercellular exchange of genetic information between
eukaryotic cells has been excluded since the publication of recent
papers reporting on nanotubes, exosomes, apoptotic bodies, and
nucleic acid – binding peptides that provide novel pathways for
cell – cell communication, with implications in health and disease
[108,109].
Functional delivery of mRNAs and micro-RNAs between mammalian cells has been demonstrated and this particular RNA was
called ‘‘exosomal shuttle RNA” (esRNA) [110] to underline its specific function. Following this new approach CNAs are now under
study for their possible function in intercellular signaling during
development [111], in epigenetic remodeling [112], tissue regeneration and fine tuning of the adaptive immune system [113]. They
may also be involved in cancer development and immune surveillance [113].
As far as cancer is concerned, nucleic acid transfer would enable
malignant cells to influence the surrounding non-malignant cells
and microenvironment in a highly specific and complex manner
to assist the tumor in nutrient supply, invasion and metastasis
[108]. Analogously, Al-Nedawi et al. [114] hypothesized that different clones of the same tumor can promote neoplastic proliferation
by the exchanging of RNA-based signals.
Researchers in this field believe that this new approach to the
understanding of cell-to-cell communication can strongly influence the way we treat various diseases [108].
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10. Conclusions
We are optimistic that circulating nucleic acid analysis will become an important tool for the clinical management of cancer patients in the near future and that it could be used successfully for
prenatal diagnosis even if, despite a great interest in this field
and ample possibilities, results are contradictory and confusing
[115].
Notwithstanding the lack of a precise knowledge on the origin
and function of CNA, many researchers are actively working on
the discovery of sensitive and specific markers in plasma/serum
based on the detection of DNA variants or anomalous RNA expression able to evidence the presence of pathological conditions, especially in neoplastic diseases and pregnancy. The involvement in
studies related to CNA derives from the conviction they represent
a non-invasive tool for disease screening and for the early detection of pathological conditions. We are confident that new analytical principles, innovative methods with extraordinary sensitivity
and specificity features, pre-analytical suitable procedures and
standardized protocols for sample collection and analysis will be
implemented in the next future. A deeper understanding of the
biology of CNA can derive from an advance in the methodological
and technological support to the study of these molecules.
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